CN117947198A - Reagent, kit, method and application for detecting bifidobacterium longum subspecies infantis BLI - Google Patents
Reagent, kit, method and application for detecting bifidobacterium longum subspecies infantis BLI Download PDFInfo
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Abstract
The invention discloses a reagent, a kit, a method and application for detecting bifidobacterium longum subspecies infancy BLI, which select a nucleotide sequence shown as SEQ ID NO.1 in the whole genome of the bifidobacterium longum subspecies infancy BLI as a detection target, and disclose a specific detection primer group and a specific probe, wherein the kit has good specificity and sensitivity, the detection speed is high, the typing detection of the bifidobacterium longum subspecies infancy BLI in a DNA sample can be realized under the constant temperature condition, and the detection sensitivity can reach the DNA concentration of 10 fg/uL.
Description
Technical Field
The invention relates to a novel target, a primer group, a probe group, a kit, a detection method and application, and belongs to the technical field of biomolecule detection.
Background
Bifidobacterium longum subsp. Infantis (Bifidobacterium longum subsp. Infantis, BLI) is an important beneficial strain commonly found in the gut flora of infants. Since the initial isolation in 1899, bifidobacteria have been shown to dominate the intestinal flora of breast-fed infants, whereas bifidobacterium longum subspecies infanti is the predominant species therein, which metabolizes human milk oligosaccharides to produce Short Chain Fatty Acids (SCFA), such as acetate, which play an important role in nutrition, intestinal and immune development, promoting direct binding to intestinal cells.
In addition to acting in the gut, SCFA produced by the bifidobacterium longum subspecies infantis BLI can also enter the circulatory system and directly affect adipose tissue, lung, brain and liver, thereby producing overall beneficial metabolic effects that benefit the host. Studies have shown that bifidobacterium longum subspecies infantis BLI are well suited for infant intestinal tract and have evolved together with maternal twins and intestinal microbiome to protect premature or term neonates and nourish healthy intestinal flora prior to weaning.
However, the problem of typing detection of bifidobacterium subspecies has remained to be solved. Therefore, a method for rapidly typing and detecting bifidobacterium subspecies is needed in clinic, and technical support is provided for the health evaluation of the intestinal tracts of infants.
Currently, the identification method of Bifidobacterium longum subspecies infantis BLI is mainly Polymerase Chain Reaction (PCR). The conventional Polymerase Chain Reaction (PCR) has been continued as a first generation PCR technique until now, and has become a "gold standard" for nucleic acid diagnosis, which has been widely used in strain detection and the like. Whereas fluorescent quantitative PCR (qPCR) is the second generation PCR technology first developed by ABI in the United states in 1996, and is classified into a dye method (SYBR Green) and a probe method (Taq Man). The qPCR technology is superior to the traditional PCR technology in specificity, sensitivity and repeatability, and the probe method can design a plurality of probes to realize multiple detection, can meet the one-time identification of a plurality of subtypes of bifidobacteria, and is widely applied to laboratory detection in a time-saving and labor-saving manner. Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification technology developed by Japanese scholars in 2000 that performs an amplification reaction by designing 4 specific primers on 6 different segments of a target DNA strand under the action of a strand displacing DNA polymerase. The method has high specificity, is simple to operate, can be completed within 1 hour only by using a constant-temperature water bath kettle, and can complete diagnosis only by using the presence or absence of an amplified product. The above nucleic acid detection techniques either require a long time or require the aid of expensive instruments, and the sensitivity is to be further improved.
The ladder-type melting temperature nucleic acid isothermal amplification technology (LMTIA) is a novel nucleic acid isothermal amplification technology, can realize stable amplification of nucleic acid within 30 minutes, and has higher sensitivity and specificity. However, LMTIA technology has not been reported in the infant intestinal bifidobacterium typing detection at present. The LMTIA technology is applied to the parting detection of the bifidobacterium longum subspecies infancy BLI, and a novel detection technology method for the bifidobacterium longum subspecies infancy BLI for laboratory diagnosis and clinical diagnosis is established, so that the method has very important practical significance.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, and provides a novel target, a primer group, a probe group, a kit, a detection method and application, and the typing detection of bifidobacterium longum subspecies infantis BLI in a DNA sample can be realized under the constant temperature condition.
In order to achieve the above purpose, the invention is realized by adopting the following technical scheme:
The first object of the invention is to provide an application of a nucleotide sequence shown as SEQ ID NO.1 as a detection target in preparing a bifidobacterium longum subspecies infancy BLI detection reagent. The bifidobacterium longum subspecies infantis BLI whole genome was selected from position 2778770 to position 2778890. The nucleotide sequence shown in SEQ ID NO.1 is:
GCGTCACTTTGAGGTCTCCTTATAAAACAAGATCTCAAGCAGTCTACCCGAAGGTAGGGAAGACGATGCTTGCAATCGTGCATGGTCCCGG。
The second object of the invention is to provide an application of a nucleotide sequence shown as SEQ ID NO.1 in preparing a bifidobacterium longum subspecies infancy BLI detection kit. The bifidobacterium longum subspecies infantis BLI whole genome was selected from position 2778770 to position 2778890. The nucleotide sequence shown in SEQ ID NO.1 is:
GCGTCACTTTGAGGTCTCCTTATAAAACAAGATCTCAAGCAGTCTACCCGAAGGTAGGGAAGACGATGCTTGCAATCGTGCATGGTCCCGG。
the third object of the present invention is to provide a reagent for detecting bifidobacterium longum subspecies infantis BLI, comprising at least a primer set and a probe;
the primer group comprises a primer one BLI-23-F1, a primer two BLI-23-B1, a primer three BLI-23-LF and a primer four BLI-23-LB;
the base sequence of the primer BLI-23-F1 is shown as SEQ ID NO. 2;
the base sequence of the primer BLI-23-B1 is shown as SEQ ID NO. 3;
the base sequence of the primer three BLI-23-LF is shown as SEQ ID NO. 4;
The base sequence of the primer tetrabll-23-LB is shown in SEQ ID No. 5;
the base sequence of the probe BLI-23-LB Pr is shown as SEQ ID NO. 6;
the probe BLI-23-LB Pr is a proofreading enzyme-mediated probe, and two ends of the probe BLI-23-LB Pr are respectively connected with a fluorescent group 6-FAM and a quenching group BHQ1.
A fourth object of the present invention is to provide a kit for detecting a bifidobacterium longum subspecies infancy BLI, comprising the above-described reagent for detecting a bifidobacterium subspecies infancy BLI.
Further, the kit further comprises Mix Buffer (2 x), bst polymerase and ddH 2 O.
A fifth object of the present invention is to provide a method for detecting bifidobacterium longum subspecies infancy BLI, implemented with a kit for detecting bifidobacterium subspecies infancy BLI; the method comprises the following steps:
Obtaining a DNA sample to be detected;
Preparing LMTIA a reaction system by using the reagent for detecting the bifidobacterium longum subspecies infantis BLI described in 4, and placing the prepared LMTIA reaction system in a real-time fluorescence PCR instrument to perform isothermal amplification on the DNA sample to be detected;
and obtaining a fluorescent signal in the real-time fluorescent PCR instrument, and judging whether positive amplification exists in the DNA sample to be detected according to the fluorescent signal.
Wherein, the step of judging whether the DNA sample to be detected has positive amplification according to the fluorescence signal comprises the following steps:
drawing an amplification result graph according to the acquired fluorescent signals;
If an amplification curve appears in the amplification result diagram, judging that positive amplification exists in the DNA sample to be detected, wherein the DNA sample to be detected contains bifidobacterium longum subspecies infancy BLI;
if the amplification curve does not appear in the amplification result, judging that the DNA sample to be detected does not have positive amplification, wherein the DNA sample to be detected does not contain bifidobacterium longum subspecies infantis BLI.
Further, the isothermal amplification temperature was 63℃and the amplification time was 20 minutes.
Further, the molar ratio of the primer one BLI-23-F1, the primer two BLI-23-B1, the primer three BLI-23-LF, the primer four BLI-23-LB and the probe BLI-23-LB Pr in the LMTIA reaction system is 4:4:1:1:5.
The sixth object of the invention is to provide a reagent for detecting the Bifidobacterium longum subspecies infancy BLI and the application of the kit for detecting the Bifidobacterium longum subspecies infancy BLI in non-disease diagnosis of the Bifidobacterium longum subspecies infancy BLI. In another aspect, the present invention provides the use of the above-described primer set and probe set for detecting bifidobacterium longum subspecies infancy BLI for non-diagnostic purposes.
Compared with the prior art, the invention has the beneficial effects that:
The primer group, the probe, the kit and the detection method for detecting the bifidobacterium longum subspecies infancy BLI have good specificity and sensitivity, the detection speed is high, and the parting detection of the bifidobacterium longum subspecies infancy BLI in the DNA sample can be realized under the constant temperature condition.
The sensitivity of the BLI primer group, the probe group, the kit and the detection method for detecting the bifidobacterium longum subspecies infantis can reach 10 fg/uL.
Drawings
FIG. 1 shows the target of the bifidobacterium longum subspecies infantis BLI of the present invention;
FIG. 2 shows amplification curves of samples of bifidobacterium longum subspecies infantis BLIDNA at different temperatures according to the present invention;
FIG. 3 shows the amplification curves of the Bifidobacterium longum subspecies infantis BLI DNA samples of the present invention;
FIG. 4 shows amplification curves of samples of BLI DNA from different concentrations of Bifidobacterium longum subspecies infantis according to the present invention.
Detailed Description
The invention is further described below with reference to the accompanying drawings. The following examples are only for more clearly illustrating the technical aspects of the present invention, and are not intended to limit the scope of the present invention.
Example 1 Bifidobacterium longum subspecies infantis BLI detection reagent and kit
According to the invention, a sequence with a ladder-shaped melting temperature is analyzed and selected through Oligo7 software, BLAST comparison analysis is carried out, and a nucleotide sequence shown as SEQ ID NO.1 is determined as a detection target. Specifically, the detection target is located at positions 2778770 to 2778890 of the bifidobacterium longum subspecies infantis BLI whole genome. The nucleotide sequence shown in SEQ ID NO.1 is:
GCGTCACTTTGAGGTCTCCTTATAAAACAAGATCTCAAGCAGTCTACCCGAAGGTAGGGAAGACGATGCTTGCAATCGTGCATGGTCCCGG。
based on the detection target, primer3Plus is used for Primer design, and a Primer group and a probe for detecting bifidobacterium longum subspecies infantis BLI are obtained through screening and synthesized by general biological systems (Anhui) limited company.
The primer group comprises a primer one BLI-23-F1, a primer two BLI-23-B1, a primer three BLI-23-LF and a primer four BLI-23-LB. Wherein the primer one BLI-23-F1 has the same nucleotide sequence as the bifidobacterium longum subspecies infantis complete genome from 2778770 th to 2778891 th and has the complementary nucleotide sequence from the bifidobacterium longum subspecies infantis complete genome from 2778811 th to 2778822 th, the primer two BLI-23-B1 has the complementary nucleotide sequence from the bifidobacterium subspecies infantis complete genome from 2778843 th to 2778860 th and has the same nucleotide sequence from the bifidobacterium subspecies infantis complete genome from 2778816 th to 2778826 th, the primer three BLI-23-LF has the complementary nucleotide sequence from the bifidobacterium subspecies infantis from 2778793 th to 2778810 th, the primer four BLI-23-LB has the same nucleotide sequence from the bifidobacterium subspecies infantis from 2778827 th to 2778842 th and the probe two BLI-23-Pr has the same nucleotide sequence from the bifidobacterium subspecies infantis from 2778827 th to 2778846 th as shown in Table 1.
TABLE 1 Bifidobacterium longum subspecies infantis BLI primer set and probe sequence
In which, as an exemplary embodiment, both ends of the probe BLI-23-LB Pr are labeled with a fluorescent group 6-FAM and a quenching group BHQ1, respectively.
A kit for detecting the BLI of the subspecies infantis of bifidobacterium longum comprises Mix Buffer (2 x), bst polymerase, a mixed solution of a primer combination and a probe, and ddH 2 O. The sources of each test agent are shown in Table 2.
TABLE 2 Experimental reagents and sources
Example 2a procedure for the detection of bifidobacterium longum subspecies infantis BLI:
Obtaining a DNA sample to be detected: the extraction of the DNA of the bifidobacterium longum subspecies infantis BLI was performed with reference to the bacterial genomic DNA/RNA rapid extraction kit instructions.
Preparing LMTIA reaction system, as shown in table 3, and placing the prepared LMTIA reaction system in a real-time fluorescence PCR instrument to perform isothermal amplification on the DNA sample to be detected;
Table 3 LMTIA reaction system (8 [ mu ] L/10 [ mu ] L)
Acquiring a fluorescent signal in a real-time fluorescent PCR instrument, and judging whether positive amplification exists in the DNA sample to be detected according to the fluorescent signal:
drawing an amplification result graph according to the acquired fluorescent signals;
if an amplification curve appears in the amplification result diagram, judging that positive amplification exists in the DNA sample to be detected, wherein the DNA sample to be detected contains bifidobacterium longum subspecies infancy BLI; if the amplification curve does not appear in the amplification result, judging that the DNA sample to be detected does not have positive amplification, wherein the DNA sample to be detected does not contain bifidobacterium longum subspecies infantis BLI.
Test example 1 determination of the isothermal amplification temperature of Bifidobacterium longum subspecies infantis BLI
And (3) taking PCR eight-joint tubes, respectively adding the prepared reaction reagents in table 3 into 16 single tubes, wherein a LMTIA reaction system does not comprise template DNA, and 8 mu L of each single tube is adopted.
2 [ Mu ] L ddH 2 O was added to 8 single tubes as a negative control. 2 [ mu ] L of bifidobacterium longum subspecies infantis BLI DNA samples are added to the other 8 single tubes.
A Gentier E full-automatic medical PCR analysis system is used for setting the temperature gradient to 59 ℃, 61 ℃, 63 ℃ and 65 ℃, collecting fluorescence signals every 30 seconds, collecting 40 fluorescence signals in total, and drawing an amplification result graph according to the obtained fluorescence signals.
Referring to FIG. 2, the Bifidobacterium longum subspecies infantis BLI DNA samples were free of primer dimer-induced non-specific amplification at 4 temperatures of 59 ℃, 61 ℃, 63 ℃ and 65 ℃. Comprehensively considering the amplification efficiency, fluorescence intensity and reproducibility of the amplification curve of the bifidobacterium longum subspecies infantis DNA sample at different temperatures, and determining the optimal temperature of the isothermal amplification at 63 ℃.
Test example 2 Bifidobacterium longum subspecies infantis BLI detection reagent specificity
Taking PCR eight-joint tubes, adding the prepared reaction reagents (the system does not comprise template DNA, 8 mu L of each single tube) in 16 single tubes respectively,
2 [ Mu ] L ddH 2 O was added to 2 single tubes as a negative control. In addition, 1 [ mu ] L of bifidobacterium longum subspecies infancy BLI DNA sample, 1 [ mu ] L of bifidobacterium subspecies longum BLL DNA sample, 1 [ mu ] L of bifidobacterium subspecies longum BLS DNA sample, 1 [ mu ] L of bifidobacterium animalis subspecies lactis BALDNA sample, 1 [ mu ] L of bifidobacterium animalis subspecies BAA DNA sample, 1 [ mu ] L of bifidobacterium breve Bbreve DNA sample and 1 [ mu ] L of bifidobacterium bifidus BBI DNA sample are respectively added into 14 single tubes, and two reactions are arranged in parallel.
The temperature was set to 63 ℃ using Gentier 96E full-automatic medical PCR analysis system, fluorescence signals were collected every 30 seconds for a total of 40 fluorescence signals, and an amplification result graph was drawn from the obtained fluorescence signals, referring to fig. 3.
Referring to FIG. 3, if a better amplification curve appears in the amplification result diagram after isothermal amplification only when the Bifidobacterium longum subspecies BLI DNA sample is added, the isothermal amplification reaction is positive; and if amplification curves do not appear in amplification result graphs corresponding to other species of source DNA samples, the isothermal amplification reactions are negative. Therefore, the established LMTIA detection method for detecting the BLI of the long bifidus subspecies infant has higher specificity.
Test example 3 sensitivity of Bifidobacterium longum subspecies infantis BLI detection reagent
Carrying out gradient dilution on a bifidobacterium longum subspecies infancy BLI DNA sample, wherein the diluted solution is TE buffer solution, and the dilution concentration is 1x10 4fg/µL、1x103fg/µL、1x102 fg/mu L, 10 fg/mu L and 1 fg/mu L.
And (3) taking PCR eight-joint tubes, respectively adding the reaction systems prepared in the table 3 into 12 single tubes, wherein the LMTIA reaction systems do not comprise template DNA, and 8 mu L of each single tube is adopted.
2 Mu L ddH 2 O is added into 2 single tubes to serve as negative control; adding 2[ mu ] L1 x10 4 fg/[ mu ] L of bifidobacterium longum subspecies infancy BLI DNA samples into the other 2 single tubes respectively; adding 2[ mu ] L1 x10 3 fg/[ mu ] L of bifidobacterium longum subspecies infancy BLI DNA samples into the other 2 single tubes respectively; adding 2[ mu ] L1 x10 2 fg/[ mu ] L of bifidobacterium longum subspecies infancy BLI DNA samples into the other 2 single tubes respectively; 2[ mu ] L10 fg/[ mu ] L of bifidobacterium longum subspecies infancy BLI DNA samples are respectively added into the other 2 single tubes; and 2[ mu ] L1 fg/[ mu ] L of bifidobacterium longum subspecies infancy BLI DNA samples are respectively added into the other 2 single tubes.
The temperature was set to 63 ℃ using Gentier 96E full-automatic medical PCR analysis system, fluorescence signals were collected every 30 seconds for a total of 40 fluorescence signals, and an amplification result graph was drawn from the obtained fluorescence signals, referring to fig. 4.
Referring to FIG. 4, when the concentration of the Bifidobacterium longum subspecies infantis BLI DNA sample is 1x10 4fg/µL、1x103fg/µL、1x102 fg/mu L and 10 fg/mu L, after isothermal amplification, a better amplification curve appears in an amplification result diagram, and the isothermal amplification reaction is positive. Therefore, the sensitivity of the bifidobacterium longum subspecies infancy BLI detection method can reach the DNA concentration of 10 fg/mu L, namely, under a reaction system of 10 mu L, the DNA concentration of the total sample of the bifidobacterium longum subspecies infancy BLIDNA reaches 10 fg/mu L can be detected.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that modifications and variations could be made by those skilled in the art without departing from the technical principles of the present invention, and such modifications and variations should also be regarded as being within the scope of the invention.
Claims (9)
1. The nucleotide sequence shown as SEQ ID NO.1 is used as a detection target in the preparation of a bifidobacterium longum subspecies infantis BLI detection reagent.
2. The nucleotide sequence shown as SEQ ID NO.1 is applied to the preparation of a bifidobacterium longum subspecies infantis BLI detection kit.
3. A reagent for detecting bifidobacterium longum subspecies infantis BLI, characterized in that: at least comprising a primer group and a probe;
the primer group comprises a primer one BLI-23-F1, a primer two BLI-23-B1, a primer three BLI-23-LF and a primer four BLI-23-LB;
the base sequence of the primer BLI-23-F1 is shown as SEQ ID NO. 2;
the base sequence of the primer BLI-23-B1 is shown as SEQ ID NO. 3;
the base sequence of the primer three BLI-23-LF is shown as SEQ ID NO. 4;
The base sequence of the primer tetrabll-23-LB is shown in SEQ ID No. 5;
the base sequence of the probe BLI-23-LB Pr is shown as SEQ ID NO. 6;
the probe BLI-23-LB Pr is a proofreading enzyme-mediated probe, and two ends of the probe BLI-23-LB Pr are respectively connected with a fluorescent group 6-FAM and a quenching group BHQ1.
4. A kit for detecting bifidobacterium longum subspecies infancy BLI, comprising the reagent for detecting bifidobacterium subspecies infancy BLI of claim 3.
5. The kit for detecting bifidobacterium longum subspecies infantis BLI according to claim 4, wherein: the kit also comprises Mix Buffer (2 x), bst polymerase and ddH 2 O.
6. The kit for detecting bifidobacterium longum subspecies infantis BLI of claim 5, wherein: the molar ratio of the primer one BLI-23-F1, the primer two BLI-23-B1, the primer three BLI-23-LF, the primer four BLI-23-LB and the probe BLI-23-LB Pr is 4:4:1:1:5.
7. A method for detecting bifidobacterium longum subspecies infancy BLI, characterized in that it is carried out using a kit for detecting bifidobacterium subspecies infancy BLI according to claim 6;
The method comprises the following steps:
Obtaining a DNA sample to be detected;
Preparing LMTIA a reaction system, and placing the prepared LMTIA reaction system in a real-time fluorescence PCR instrument to perform isothermal amplification on the DNA sample to be detected; the isothermal amplification temperature is 63 ℃ and the amplification time is 20 minutes;
and obtaining a fluorescent signal in the real-time fluorescent PCR instrument, and judging whether positive amplification exists in the DNA sample to be detected according to the fluorescent signal.
8. The method for detecting bifidobacterium longum subspecies infantis BLI as in claim 7, wherein said step of determining whether a positive amplification is present in the DNA sample to be detected based on the fluorescent signal comprises:
drawing an amplification result graph according to the acquired fluorescent signals;
If an amplification curve appears in the amplification result diagram, judging that positive amplification exists in the DNA sample to be detected, wherein the DNA sample to be detected contains bifidobacterium longum subspecies infancy BLI;
if the amplification curve does not appear in the amplification result, judging that the DNA sample to be detected does not have positive amplification, wherein the DNA sample to be detected does not contain bifidobacterium longum subspecies infantis BLI.
9. Use of a reagent for detecting bifidobacterium longum subspecies infantis BLI according to claim 3, or a kit for detecting bifidobacterium subspecies infantis BLI according to any one of claims 4 to 5, in the non-disease diagnosis of bifidobacterium subspecies infantis BLI.
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